Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself.

Structural studies of the 5'-phenazinium-tethered matched and G-A-mismatched DNA duplexes by NMR spectroscopy.

The mechanism through which modified oligo-DNA analogues act as antisense repressors at the transcriptional and translational level of gene expression is based on the information content in the nucleotide sequence which is determined by the specific base pairing. The efficiency of such action is largely determined by the stability of the duplex formed between the oligonucleotide reagent and the target sequence and also by the mismatched base pairing, such as G-A, that occurs during replication or recombination. We herein report that the phenazinium (Pzn)-tethered matched duplex p(d(TGTTTGGC)):(Pzn)-p(d(CCAAACA)) (III) (Tm = 50 degrees C) has a much larger stability than the parent matched duplex p(d(TGTTTGGC)):p(d(CCAAACA)) (I) (Tm = 30 degrees C). On the other hand, the Pzn-tethered G-A-mismatched duplex p(d(TGTTTGGC)):(Pzn)-p(d(ACAAACA)) (IV) (Tm = 34 degrees C) is only slightly more stable than its parent mismatched duplex p(d(TGTTTGGC)):p(d(ACAAACA)) (Tm = 25 degrees C). A detailed 500 MHz NMR study and constrained MD refinements of NMR-derived structures have been undertaken for the DNA duplexes (I), (II), (III) and (IV) in order to understand the structural basis of stabilization of Pzn-tethered matched DNA duplex (delta Tm = 20 degrees C) compared to mismatched duplex (delta Tm = 9 degrees C). Assignment of the 1H-NMR (500 MHz) spectra of the duplexes has been carried out by 2D NOESY, HOHAHA and DQF-COSY experiments. The torsion angles have been extracted from the J-coupling constants obtained by simulation of most of the DQF-COSY cross-peaks using program SMART. The solution structure of the duplexes were assessed by an iterative hybride relaxation matrix method (MORASS) combined with NOESY distances and torsion angles restrained molecular dynamics (MD) using program Amber 4.0. The standard Amber 4.0 force-field parameters were used for the oligonucleotide in conjunction with the new parameters for Pzn residue which was obtained by full geometry optimization using ab initio program (3-21G basis set). It has been shown that mismatched G-A bases are in the anti-anti conformation. The mismatched 7G-1A form stable base pairs through inter-strand hydrogen bonds (N7(A)...HN2(G) (1.92 A) with a subtended angle of 176 degrees and N3(G)...HN6(A) (2.01 A) with a subtended angle of 153 degrees (the 'amino-type' hydrogen bond)) and a propeller twist of 36 degrees for 7G-1A residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of bleomycin A5 oligonucleotide derivatives and site-specific cleavage of the DNA target.

A method for coupling bleomycin A5 to oligonucleotides is proposed. The reaction was carried out between an amino group of a spermidine residue of the Cu(II) complex of bleomycin A5 (Cu(II)Blm-RH) and a 5'-phosphate group of the oligonucleotides d(pCCAAACA) (I), d(pCGTCCTC) (II), d(pT)16 (III), d(pCAAACA) (IV), and d(pGCCAAACA) (V) activated with a mixture of triphenylphosphine and 2,2'-dipyridyl disulfide in the presence of 4-(N,N-dimethylamino)pyridine 1-oxide. The yields of the products Cu(II)Blm-R-d(pCCAAACA) (Ia), Cu(II)Blm-R-d(pCGTCCTC) (IIa), Cu(II)Blm-R-d(pT)16 (IIIa), Cu(II)Blm-R-d(pCAAACA) (IVa), and Cu(II)Blm-R-d(pGCCAAACA) (Va) were 60-80%. After removal of the Cu(II) ion from the bleomycin A5 oligonucleotide derivatives Ia-IIIa, compounds Ib-IIIb were obtained. Compounds Ia, IVa, Va, and Ib-IIIb were further used for modification of the target d(pTGTTTGGCGAAGGA) in the presence of Fe(II) ions and 2-mercaptoethanol. Site-specific cleavage of the target by Blm coupled to complementary oligonucleotides was demonstrated. It was shown that efficiency and position of cleavage of the complementary reagents Ia, Ib, IVa, and Va are determined by their oligonucleotide part while the action of thenoncomplementary reagents IIb and IIIb was similar to that of the free antibiotic.

Direct cleavage of a DNA fragment by a bleomycin-oligonucleotide derivative.

Bleomycin A5 oligonucleotide derivative was used for direct cleavage of a DNA target. In the presence of Fe2+ ions and 2-mercaptoethanol, Blm-R-pd(CCAAACA) (I) damaged the target. pd(TGTTTGGCGAAGGA), with the yield of 80% , without affecting its own oligonucleotide tail. The sites of cleavage were T3-T5 and G6-G7. Unbound bleomycin A5 damaged the G6-G7-C8 site. Reagent I formed more stable complementary complexes with the target than parent oligonucleotide (ATm = 11 C).